Several naturally occurring mutations in the lipid binding domain of apoAI are associated with a decreased HDL concentration but paradoxically are not associated with premature cardiovascular disease. One such mutation is the Cys for Arg substitution at amino acid 173, which occur in apoAI-M(ilano). The major aim of the present proposal is to create an animal model in which to study the anti-atherogenic potential of apoAI-M. A single copy, homologous recombination strategy will be employed to replace the mouse apoAI gene with the human apoAI or the apoAI-M gene to create mice having equivalent expression of human apoAI or apoAIM expression. The gene targeting procedure will be carried out using ES cells from strain C57BL/6, an atherosclerosis susceptible strain, in order to examine whether the apoAI-M replacement animals are protected from dietary induced atherosclerosis. The mice expressing the human apoAI will serve as controls in studying the mechanism of any protective effect of apoAI-M. The applicant will test the following hypotheses. (1) in the background of the atherosclerosis susceptible C57BL/6 mouse on an atherogenic diet, the apoAIM gene will be more protective against lesion development than the normal human apoAI. (2) the apoAI/apoAI-M genotype in "knock-in" mice will recapitulate the unusual lipid and lipoprotein profile found in humans, since apoAI-M had only been observed in heterozygous carriers. (3) apoAI-M HDL and/or a specific sub-population of variant HDL are significantly more efficient than normal apoAI in stimulating cholesterol efflux from mouse peritoneal macrophages, thus decreasing lipid accumulation in the artery wall. The second phase of the experiments are to elucidate whether apoAI-M dimerization on HDL particles produced by disulfide bond formation is linked with apoAIM's putative protective properties. An Arg-173 to Leu mutation will be introduced into mice in the same fashion as was used for apoAI-M. Other Arg-Cys substitutions in the lipid binding domains of apoAI will be produced to determine whether these substitutions, as a class, have the potential for being protective. These studies provide a novel approach for addressing the important human issues whether apoAI-M protects against atherosclerosis and whether other Arg-Cys substitutions have the same potential.